Detection of Natural Resistance-Associated Substitutions by Ion Semiconductor Technology in HCV1b Positive, Direct-Acting Antiviral Agents-Naïve Patients

Abstract

Naturally occurring resistance-associated substitutions (RASs) can negatively impact the response to direct-acting antivirals (DAAs) agents-based therapies for hepatitis C virus (HCV) infection. Herein, we set out to characterize the RASs in the HCV1b genome from serum samples of DAA-naïve patients in the context of the SINERGIE (South Italian Network for Rational Guidelines and International Epidemiology, 2014) project. We deep-sequenced the NS3/4A protease region of the viral population using the Ion Torrent Personal Genome Machine, and patient-specific majority rule consensus sequence summaries were constructed with a combination of freely available next generation sequencing data analysis software. We detected NS3/4A protease major and minor variants associated with resistance to boceprevir (V36L), telaprevir (V36L, I132V), simeprevir (V36L), and grazoprevir (V36L, V170I). Furthermore, we sequenced part of HCV NS5B polymerase using Sanger-sequencing and detected a natural RAS for dasabuvir (C316N). This mutation could be important for treatment strategies in cases of previous therapy failure.

Structural Modeling of New Polymorphism Clusters of HCV Polymerase Isolated from Direct‐Acting Antiviral Naïve Patients: Focus on Dasabuvir and Setrobuvir Binding Affinity

“…Overall the median baseline RNA viral load of samples was 3,207,895 IU/mL, lower limit 1,030,000 IU/mL and higher limit 7,930,000 IU/mL in baseline samples (Table ). In NS5B consensus sequences, we identified several non‐synonymous substitutions, even though important AA resistance positions (i. e. 159, 368, 411, 414, 448, 553, 554, 556) were not amplified . The 316 substitution at RAS position is involved in multiple DAAs‐containing regimens, targeting Palm I region ,.…”

“…The huge variability between HCV types, at the nucleotide level, could impact the effectiveness of DAA drugs . Newly NS5B sequences were screened for non‐synonymous substitution to polymerase inhibitors within the dominant viral population . The NS5B resistance profile was characterized by direct sequencing.…”

Real‐life 3D therapy failure: Analysis of NS5A 93H RAS plus 108 K polymorphism in complex with ombitasvir by molecular modeling

“…Then, RASs could appear post‐treatment, after selection of pre‐existing substitutions among quasispecies or as novel mutations . By the way, our previous reported approach, using the Ion Personal Genome Machine (PGM) Sequencer, should be useful to check NS5A 93H RAS in minor population with a cut‐off from 1% to 15% …”

Clinical, Virological Characteristics, and Outcomes of Treatment with Sofosbuvir/Ledipasvir in Two Pediatric Patients Infected by HCV Genotype 4

“…Subtyping was performed by Versant HCV genotype v2.0 assay (LiPA) (Siemens, Healthcare Diagnostic Inc., Tarrytown, NY, USA). Fibrosis stage was estimated by transient elastometry (FibroScan ® , Echosens, Paris, France), interpreted as in References [10,12,13], and abdomen ultrasound was performed at baseline and after the end of treatment.…”